Subcellular mapping of living cells via synchrotron microFTIR and ZnS hemispheres.

FTIR imaging is a label-free, non-destructive method valuably exploited in the study of the biological process in living cells. However, the long wavelength/low spatial resolution and the strong absorbance of water are still key constrains in the application of IR microscopy ex vivo. In this work, a new retrofit approach based on the use of ZnS hemispheres is introduced to significantly improve the spatial resolution on live cell FTIR imaging. By means of two high refractive index domes sandwiching the sample, a lateral resolution close to 2.2 µm at 6 µm wavelength has been achieved, i.e. below the theoretical diffraction limit in air and more than twice the improvement (to ~λ/2.7) from our previous attempt using CaF2 lenses. The ZnS domes also allowed an extended spectral range to 950 cm-1, in contrast to the cut-off at 1050 cm-1 using CaF2. In combination with synchrotron radiation source, microFTIR provides an improved signal-to-noise ratio through the circa 12 µm thin layer of medium, thus allowing detailed distribution of lipids, protein and nucleic acid in the surround of the nucleus of single living cells. Endoplasmic reticula were clearly shown based on the lipid ν(CH) and ν(C=O) bands, while the DNA was imaged based on the ν(PO2-) band highlighting the nucleus region. This work has also included a demonstration of drug (doxorubicin) in cell measurement to highlight the potential of this approach. Graphical abstract.

Increased optical pathlength through aqueous media for the infrared microanalysis of live cells.

The study of live cells using Fourier transform infrared spectroscopy (FTIR) and FTIR microspectroscopy (FT-IRMS) intrinsically yields more information about cell metabolism than comparable experiments using dried or chemically fixed samples. There are, however, a number of barriers to obtaining high-quality vibrational spectra of live cells, including correction for the significant contributions of water bands to the spectra, and the physical stresses placed upon cells by compression in short pathlength sample holders. In this study, we present a water correction method that is able to result in good-quality cell spectra from water layers of 10 and 12 µm and demonstrate that sufficient biological detail is retained to separate spectra of live cells based upon their exposure to different novel anti-cancer agents. The IR brilliance of a synchrotron radiation (SR) source overcomes the problem of the strong water absorption and provides cell spectra with good signal-to-noise ratio for further analysis. Supervised multivariate analysis (MVA) and investigation of average spectra have shown significant separation between control cells and cells treated with the DNA cross-linker PL63 on the basis of phosphate and DNA-related signatures. Meanwhile, the same control cells can be significantly distinguished from cells treated with the protein kinase inhibitor YA1 based on changes in the amide II region. Each of these separations can be linked directly to the known biochemical mode of action of each agent. Graphical abstract.

Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy.

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells. Graphical abstract FTIR microspectroscopy is able to differentiate between mycoplasma infected cells (LC for low contamination and HC for high contamination) and control non-infected cells (CN).

Synchrotron FTIR mapping of mineralization in a microfluidic device.

Fourier transform infrared micro-spectroscopy provides an effective means of performing rapid, non-destructive, and label-free analysis of specimens according to their vibrational modes. However, as water absorbs very strongly in the infrared region, analysis of aqueous solutions in transmission mode can suffer from problems with signal saturation. We here describe the fabrication of a novel microfluidic device that overcomes this problem. Devices with channel depths of just 3 µm were constructed from calcium fluoride using photolithography and hot embossing bonding, where calcium fluoride was selected due to its transparency in the IR region. The utility of this device was then demonstrated by employing it to follow the precipitation pathways of calcium sulfate and calcium carbonate using synchrotron FTIR micro-spectroscopy. Importantly, due to the high brightness provided by synchrotron radiation, and the fact that the reacting ions (HCO3-, CO32- and SO42-) and the different mineral polymorphs all have finger print spectra in the measured IR range, this method can be used to acquire time-resolved, hyperspectral maps of the mineral particles formed within the sample cell, and then study the interaction and evolution of particles. The data provide new insight into the formation pathway of a population of crystals in confined volumes, and demonstrate that this in situ, real-time detection system provides a powerful tool for studying crystallization processes.

Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control.

We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s(-1), and therefore, fast experimental triggers are needed in order to study subturnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment, this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 µm(2). We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control.

Infrared imaging of small molecules in living cells: from in vitro metabolic analysis to cytopathology.

A major topic in InfraRed (IR) spectroscopic studies of living cells is the complexity of the vibrational spectra, involving hundreds of overlapping absorption bands from all the cellular components present at detectable concentrations. We focus on the relative contribution of both small-molecule metabolites and macromolecules, while defining the spectroscopic properties of cells and tissue in the middle IR (midIR) region. As a consequence, we show the limitations of current interpretative schemes that rely on a small number of macromolecules for IR band assignment. The discussion is framed specifically around the glycolytic metabolism of cancer cells because of the potential pharmacological applications. Several metabolites involved in glycolysis by A549 lung cancer cells can be identified by this approach, which we refer to as Correlated Cellular Spectro-Microscopy (CSM). It is noteworthy that the rate of formation or consumption of specific molecules could be quantitatively assessed by this approach. We now extend this analysis to the two-dimensional case by performing IR imaging on single cells and cell clusters, detecting variations of metabolite concentration in time and space across the sample. The molecular detail obtained from this analysis allows its use in evaluating the pharmacological effect of inhibitors of glycolytic enzymes with potential consequences for in vitro drug testing. Finally we highlight the implications of the spectral contribution from cellular metabolites on applications in IR spectral cytopathology (SCP).

Broadband near-field infrared spectromicroscopy using photothermal probes and synchrotron radiation.

In this paper, we experimentally demonstrate the use of infrared synchrotron radiation (IR-SR) as a broadband source for photothermal near-field infrared spectroscopy. We assess two methods of signal transduction; cantilever resonant thermal expansion and scanning thermal microscopy. By means of rapid mechanical chopping (50-150 kHz), we modulate the IR-SR at rates matching the contact resonance frequencies of atomic force microscope (AFM) cantilevers, allowing us to record interferograms yielding Fourier transform infrared (FT-IR) photothermal absorption spectra of polystyrene and cyanoacrylate films. Complementary offline measurements using a mechanically chopped CW IR laser confirmed that the resonant thermal expansion IR-SR measurements were below the diffraction limit, with a spatial resolution better than 500 nm achieved at a wavelength of 6 µm, i.e. λ/12 for the samples studied. Despite achieving the highest signal to noise so far for a scanning thermal microscopy measurement under conditions approaching near-field (dictated by thermal diffusion), the IR-SR resonant photothermal expansion FT-IR spectra measured were significantly higher in signal to noise in comparison with the scanning thermal data.

Discrimination between two different grades of human glioma based on blood vessel infrared spectral imaging.

Gliomas are brain tumours classified into four grades with increasing malignancy from I to IV. The development and the progression of malignant glioma largely depend on the tumour vascularization. Due to their tissue heterogeneity, glioma cases can be difficult to classify into a specific grade using the gold standard of histological observation, hence the need to base classification on a quantitative and reliable analytical method for accurately grading the disease. Previous works focused specifically on vascularization study by Fourier transform infrared (FTIR) spectroscopy, proving this method to be a way forward to detect biochemical changes in the tumour tissue not detectable by visual techniques. In this project, we employed FTIR imaging using a focal plane array (FPA) detector and globar source to analyse large areas of glioma tumour tissue sections via molecular fingerprinting in view of helping to define markers of the tumour grade. Unsupervised multivariate analysis (hierarchical cluster analysis and principal component analysis) of blood vessel spectral data, retrieved from the FPA images, revealed the fine structure of the borderline between two areas identified by a pathologist as grades III and IV. Spectroscopic indicators are found capable of discriminating different areas in the tumour tissue and are proposed as biomolecular markers for potential future use of grading gliomas. Graphical Abstract Infrared imaging of glioma blood vessels provides a means to revise the pathologists' line of demarcation separating grade III (GIII) from grade IV (GIV) parts.

Optimal Sample Preparation for the Analysis of Micrometric Heterogeneous Samples.

Precise microanalytical techniques are essential in many fields such as cultural heritage materials, showing complex layered microstructures containing a wide range of materials of diverse nature and hardness. Noninvasive sample manipulation and preparation is required to avoid, as much as possible, sample contamination, which may strongly limit the materials identification. The method proposed consists in the application of thin gold or carbon protecting layers before embedding the samples in synthetic resin for microtoming. The validity and optimal procedure is checked for those materials most often found on the surface of paintings: varnishes (natural resins and wax). An artwork sample is similarly prepared and analyzed by optical microscopy (OM), scanning electron microscopy (SEM/EDS), micro-infrared spectroscopy (µFTIR/µSR-FTIR), and X-ray diffraction (µSR-XRD) with synchrotron light.

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.

Probing single-tumor cell interactions with different-age type I collagen networks by synchrotron-based Fourier transform infrared microspectroscopy.

We report here on a first study using synchrotron radiation-based Fourier transform infrared microspectroscopy and imaging to investigate HT1080 human fibrosarcoma cells grown onto different-aged type I collagen networks. Spectral images were analyzed with k-means and fuzzy C-means (FCM) clustering algorithms. K-means delineated tumor cells from their surrounding collagen networks and the latter as a function of age mainly due to specific changes in the sugar absorption region. The FCM analysis gave a better nuance of the spectral images. A progression of the biochemical information was observed upon going from the cellular compartments to the pericellular contact regions and to the intact collagens of the different age groups. Two spectral markers based on sugar and protein bands via the intensity ratio (I1032/I1655) and band area ratio (Asugar/Aamide II), showed an increase in advanced glycation endproducts (AGEs) with age. A clear-separation of the three age groups was obtained for spectra originating from the peripheral contact areas mainly due to changes in protein band intensities. The above-described markers decreased to constant levels for the three conditions indicating a masking of the biochemical information. These results hold promises to better understand the impact of age on tumor progression processes while highlighting new markers of the tumor cell invasion front.

Synchrotron based infrared imaging and spectroscopy via focal plane array on live fibroblasts in D2O enriched medium.

We successfully tested the viability of using synchrotron-based full-field infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information.

Rapid recognition of drug-resistance/sensitivity in leukemic cells by Fourier transform infrared microspectroscopy and unsupervised hierarchical cluster analysis.

We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 µm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.

The effect of optical substrates on micro-FTIR analysis of single mammalian cells.

The study of individual cells with infrared (IR) microspectroscopy often requires living cells to be cultured directly onto a suitable substrate. The surface effect of the specific substrates on the cell growth-viability and associated biochemistry-as well as on the IR analysis-spectral interference and optical artifacts-is all too often ignored. Using the IR beamline, MIRIAM (Diamond Light Source, UK), we show the importance of the substrate used for IR absorption spectroscopy by analyzing two different cell lines cultured on a range of seven optical substrates in both transmission and reflection modes. First, cell viability measurements are made to determine the preferable substrates for normal cell growth. Successively, synchrotron radiation IR microspectroscopy is performed on the two cell lines to determine any genuine biochemically induced changes or optical effect in the spectra due to the different substrates. Multivariate analysis of spectral data is applied on each cell line to visualize the spectral changes. The results confirm the advantage of transmission measurements over reflection due to the absence of a strong optical standing wave artifact which amplifies the absorbance spectrum in the high wavenumber regions with respect to low wavenumbers in the mid-IR range. The transmission spectra reveal interference from a more subtle but significant optical artifact related to the reflection losses of the different substrate materials. This means that, for comparative studies of cell biochemistry by IR microspectroscopy, it is crucial that all samples are measured on the same substrate type.

Isolating stem cells in the inter-follicular epidermis employing synchrotron radiation-based Fourier-transform infrared microspectroscopy and focal plane array imaging.

Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ = 2.5-25 µm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell's inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IR-derived markers. Paraffin-embedded samples of human scalp skin (n = 4) were obtained, and 10-µm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15 × 15 µm) and also imaged employing globar-source FTIR focal plane array (FPA) imaging (5.4 × 5.4 µm). Dependent on the location of derived spectra, wavenumber-absorbance/intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO(2)(-) vibrational modes (1,225 and 1,080 cm(-1)), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO (2) (-) vibrational modes as potential putative SC markers.

Electric field standing wave artefacts in FTIR micro-spectroscopy of biological materials.

FTIR absorption micro-spectroscopy is a widely used, powerful technique for analysing biological materials. In principle it is a straightforward linear absorption spectroscopy, but it can be affected by artefacts that complicate the interpretation of the data. In this article, artefacts produced by the electric-field standing-wave (EFSW) in micro-reflection-absorption (transflection) spectroscopy are investigated. An EFSW is present at reflective metallic surfaces due to the interference of incident and reflected light. The period of this standing wave is dependent on the wavelength of the radiation and can produce non-linear changes in absorbance with increasing sample thickness (non-Beer-Lambert like behaviour). A protein micro-structure was produced as a simple experimental model for a biological cell and used to evaluate the differences between FTIR spectra collected in transmission and transflection. By varying the thickness of the protein samples, the relationship between the absorbance and sample thickness in transflection was determined, and shown to be consistent with optical interference due to the EFSW coupled with internal reflection from the sample top surface. FTIR spectral image data from MCF 7 breast adenocarcinoma cells was then analysed to determine the severity of the EFSW artefact in data from a real sample. The results from these measurements confirmed that the EFSW artefact has a profound effect on transflection spectra, and in this case the main spectral variations were related to the sample thickness rather than any biochemical differences.

First Quantitative Imaging of Organic Fluorine within Angiogenic Tissues by Particle Induced Gamma-Ray Emission (PIGE) Analysis: First PIGE Organic Fluorine Imaging.

PET (Positron Emission Tomography) allows imaging of the in vivo distribution of biochemical compounds labeled with a radioactive tracer, mainly 18F-FDG (2-deoxy-2-[18F] fluoro-D-glucose). 18F only allows a relatively poor spatial resolution (2-3 mm) which does not allow imaging of small tumors or specific small size tissues, e.g. vasculature. Unfortunately, angiogenesis is a key process in various physiologic and pathologic processes and is, for instance, involved in modern anticancer approaches. Thus ability to visualize angiogenesis could allow early diagnosis and help to monitor the response of cancer to specific chemotherapies. Therefore, indirect analytical techniques are required to assess the localization of fluorinated compounds at a micrometric scale. Multimodality imaging approaches could provide accurate information on the metabolic activity of the target tissue. In this article, PIGE method (Particle Induced Gamma-ray Emission) was used to determine fluorinated tracers by the nuclear reaction of 19F(p,p'γ)19F in tissues. The feasibility of this approach was assessed on polyfluorinated model glucose compounds and novel peptide-based tracer designed for angiogenesis imaging. Our results describe the first mapping of the biodistribution of fluorinated compounds in both vascularized normal tissue and tumor tissue.

Differentiation between normal and tumor vasculature of animal and human glioma by FTIR imaging.

Malignant gliomas are very aggressive tumors, highly angiogenic and invading heterogeneously the surrounding brain parenchyma, making their resection very difficult. To overcome the limits of current diagnostic imaging techniques used for gliomas, we proposed using FTIR imaging, with a spatial resolution from 6 to 10 µm, to provide molecular information for their histological examination, based on discrimination between normal and tumor vasculature. Differentiation between normal and tumor blood vessel spectra by hierarchical cluster analysis was performed on tissue sections obtained from xenografted brain tumors of Rag-gamma mice 28 days after intracranial implantation of glioma cells, as well as for human brain tumors obtained in clinics. Classical pathological examination and immunohistochemistry were performed in parallel to the FTIR spectral imaging of brain tissues. First on the animal model, classification of FTIR spectra of blood vessels could be performed using spectral intervals based on fatty acyl (3050-2800 cm(-1)) and carbohydrate (1180-950 cm(-1)) absorptions, with the formation of two clusters corresponding to healthy and tumor parts of the tissue sections. Further data treatments on these two spectral intervals provided interpretable information about the molecular contents involved in the differentiation between normal and tumor blood vessels, the latter presenting a higher level of fatty acyl chain unsaturation and an unexpected loss of absorption from osidic residues. This classification method was further successfully tested on human glioma tissue sections. These findings demonstrate that FTIR imaging could highlight discriminant molecular markers to distinguish between normal and tumor vasculature, and help to delimitate areas of corresponding tissue.

FT-IR spectral imaging of blood vessels reveals protein secondary structure deviations induced by tumor growth.

Vascular basement membrane remodeling is involved in tumor angiogenesis to enable tumor invasion and growth. FT-IR spectral imaging was used to determine changes in tumor blood vessels to reveal protein secondary structure in Rag-gamma immuno-deficient mice sacrificed 14 and 21 days after subcutaneous glioma implantation. For the oldest blood capillaries (diameter >20 microns), tumor growth induced a decrease in triple-helix content (1638 cm(-1); -7.3%; P < 0.05) and an increase in beta turns (1666 and 1615 cm(-1); +4%; P < 0.01). These protein-structure alterations, mainly from type IV collagen, reflected the high angiogenic stress of growing tumors. We propose to use these molecular markers of vascular basement membrane protein alterations for gradation of solid tumors by FT-IR spectral imaging.

Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue.

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700-1,480 cm(-1) spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and alpha-helix, but lower amounts of beta-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues.